Cytoenzymology is the study of enzymes as they function on and within cells. Dead or metabolically inactive cells can have as little as approximately one-quarter the enzymatic activity of living cells, Watson, J., “Enzyme Kinetic Studies in Cell Population Using Fluorogenic Substrates and Flow Cytometric Techniques”, Cytometry, 1(2), p. 143 (1980). Further, because enzymes are frequently bound in highly organized enzyme pathways, the activity of enzymes can reveal cell disruption or death.
Previously, the study of enzymatic activity within cells has been pursued primarily by three methods. The first such method determines enzyme activity by studying extra-cellular events, such as the presence or lack of the products of enzyme activity (see. e.g., Warrington, et al., U.S. Pat. No. 4,940,659; Short, et al., U.S. Pat. No. 6,174,673, etc.).
In the second such method, the cell membrane is ruptured in order to create a cytosol of cellular components including the enzyme that is the object of study. Various tests may then be performed, using either the cytosol or the purified enzyme in order to determine the activity of the enzyme. One such approach involves incubating the cell in culture medium containing high concentrations of sucrose or polyethylene glycol in order to break cellular membranes and release cytosol (Rechsteiner, M. “Osmotic lysis of pinosomes,” Methods Enzymol 1987;149:42–48; Okada, C. Y. et al., “Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles,” Cell 1982 May;29(1):33–41; Park, R. D. et al., “Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments,” J Cell Physiol 1988 June;135(3):443–450). A second such test is to provide a substrate, that is recognized by the enzyme, with a fluorescent compound which will undergo a detectable change when the substrate, or “leaving group”, is cleaved from the compound by the enzyme (see, for example Mangel et al., U.S. Pat. Nos. 4,557,862 and 4,640,893, which disclose rhodamine 110-based derivatives as fluorogenic substrates for proteinases). G. Rothe et al., Biol. Chem. Hoppe-Seyler, 373, 544–547 (1992) describe the analysis of proteinase activities using the substituted peptide-rhodamine 110 derivatives of Mangel et al. Moreover, G. Valet et al, Ann NY Acad Sci, 667, 233–251 (1993), disclose the study of white cell and thrombocyte disorders with the rhodamine 110 derivatives of Mangel et al. The methods of Rothe and Valet have been used to conduct cytoenzymological studies on the activity of enzymes with cells, but the compounds utilized by Rothe and Valet are not suitable for the study of the activity of intracellular enzymes in vital cells. The Mangel et al. compounds cannot be efficiently solubilized and transmitted through the cell membrane in a manner which will produce a reliable assay. I. Mononen, et al., Clin. Chem., 40(3), 385–388 (1994), describe the enzymatic diagnosis of aspartylglycos-aminuria by the fluorometric assay of glycosylasparaginase in serum, plasma, and lymphocytes. The study was conducted on cytosols, and not whole cells, and utilized an asparagine-substituted 7-amino-4-methylcoumarin. U.S. Pat. No. 5,070,012 to Nolan et al., describes a method of monitoring cells and trans-acting transcription elements. This method, however, is not designed for the monitoring of enzymes which are endogenous to the cell being tested.
Lucas, et al. (U.S. Pat. No. 5,698,411) and Landrum et al. (U.S. Pat. No. 5,976,822) describe the third general method for assaying enzyme activity. In this method, the enzyme activity of metabolically active whole cell s is determined. The assay employs reagents that comprise at least one water soluble assay compound having the ability to pass through a cell membrane or a water soluble physiologically acceptable salt thereof having the ability to pass through a cell membrane. The assay compound has (i) a leaving group selected so that it may be cleaved by an enzyme to be analyzed and (ii) a fluorogenic indicator group selected for its ability to have a non-fluorescent first state when joined to the leaving group, and a fluorescent second state excitable at a convenient wavelength (e.g., a wavelength above 450 nm) when the leaving group is cleaved from the indicator group by the enzyme.
Landrum et al. (U.S. Pat. No. 5,976,822) discloses assay reagents for determining the activity of an enzyme in a metabolically active whole cell, in which the assay reagent comprises at least one water soluble physiologically acceptable salt having the ability to pass through a cell membrane. The assay compound has an unblocked leaving group selected for cleavage by an enzyme to be analyzed (such as a cysteine protease (especially a caspase enzyme or a granzyme of cysteine proteases), dipeptyl peptidase and calpain), and a fluorogenic indicator group selected for its ability to have a non-fluorescent first state when joined to the leaving group, and a fluorescent second state excitable at a wavelength when the unblocked leaving group is cleaved from the indicator group by the enzyme. Various indicator groups are disclosed (4′(5′)aminorhodamine 110, 4′(5′)carboxyrhodamine 110, 4′(5′)chlororhodamine 110, 4′(5′)methylrhodamine 110, 4′(5′)sulforhodamine 110, 4′(5′)aminorhodol, 4′(5′)carboxyrhodol, 4′(5′)chlororhodol, 4′(5′)methylrhodol, 4′(5′)sulforhodol, 4′(5′)aminofluorescein, 4′(5′)carboxyfluorescein, 4′(5′)chlorofluorescein, 4′(5′)methylfluorescein, and 4′(5′)sulfofluorescein).
Such enzyme assays are particularly desirable for determining the activity of enzymes associated with tumor cell progression or apoptosis. Apoptosis, or programmed cell death, is a process that involves the activation of a genetic program when cells are no longer needed or have become seriously damaged. This process, occurring in most cells from higher eukaryotes, is necessary for normal development and maintenance of homeostasis. It is a major defense mechanism of the body, permitting the body to eliminate unwanted and possibly dangerous cells such as virus-infected cells, tumor cells, and self-reactive lymphocytes.
Apoptosis involves a cascade of specific biochemical events, and its regulation involves a large number of enzymes. These can be classified into three general categories. The first is made up of enzymes whose primary function is to suppress apoptosis. This group includes some members of the bcl-2 family. Other members of the bcl-2 family can promote apoptosis. The second group includes the intermediate genes upstream such as Fas/Fas ligand, myc, p53, and WAF1. Fas is a cell surface protein that triggers apoptosis in a variety of cell types. The Fas death pathway can be triggered by either anti-Fas monoclonal antibody or by cell-associated Fas ligand. This protein is identical to the CD95 protein. CD95 is involved in regulation of tissue development and homeostasis. Cloning of Fas and APO-1 cDNA has demonstrated that these two genes are identical. The Fas antigen is a cell surface protein that belongs to the tumor necrosis factor/nerve growth factor receptor family.
The last group includes genes, such as cysteine proteases that act as effectors of apoptosis. An example is the interleukin-1β converting enzyme (ICE) family of genes. Several homologues of ICE have been identified, including CPP32- and Ich-1-like proteases. Specific inhibitors of ICE-like proteases can inhibit apoptosis. This indicates there is a requirement for specific degradation by proteases in mammalian apoptosis. The ICE family of cysteine proteases has an indispensable role in the regulation of apoptosis. It appears that the ICE family of proteases process themselves and each other by proteolytically cleaving a “pre” enzyme into the active form. The ICE family of proteases is generically referred to as “caspase” enzymes (Alnemri, et al., Cell, Volume 87, page 171, 1996. Caspases are also thought to be crucial in the development and treatment of cancer. The “c” is intended to reflect a cysteine protease mechanism and “aspase” refers to their ability to cleave after aspartic acid, the most distinctive catalytic feature of this protease family).
Caspase proteases are reviewed by Darzynkiewicz, Z. et al. (“Flow cytometry in analysis of cell cycle and apoptosis,” Semin Hematol. 2001 April;38(2):179–93); Riss, T. L. (“Apoptosis as a biomarker in chemoprevention trials,” Urology. 2001 April;57(4 Suppl 1):141–2); Saraste, A. et al. (“Morphologic and biochemical hallmarks of apoptosis,” Cardiovasc Res. 2000 February;45(3):528–37); Akao, Y. et al. (“Arsenic-induced apoptosis in malignant cells in vitro,” Leuk Lymphoma. 2000 March;37(1–2):53–63); Bannerman, D. D. et al. (“Direct effects of endotoxin on the endothelium: barrier function and injury,” Lab Invest. 1999 October;79(10):1181–99); Saraste, A. et al. (“Morphologic criteria and detection of apoptosis,” Herz. 1999 May;24(3):189–95); Levy-Strumpf, N. et al. (“Death associated proteins (DAPs): from gene identification to the analysis of their apoptotic and tumor suppressive functions,” Oncogene. 1998 December 24;17(25):3331–40); Warner, C. M. et al. (“Role of the Ped gene and apoptosis genes in control of preimplantation development,” J Assist Reprod Genet. 1998 May;15(5):331–7); Tocci, M. J. (“Structure and function of interleukin-1 beta converting enzyme,” Vitam Horm. 1997;53:27–63); Miller, D. K. et al. (“The IL-1 beta converting enzyme as a therapeutic target,” Ann NY Acad Sci. 1993 Nov. 30;696:133–48). Zhang, et al. (U.S. Pat. No. 6,248,904) discusses fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof
Despite all such advances, a need exists for improved reagents for use in cytoenzymology, and particularly for improved reagents that can be used to assay enzymes associated with tumor cell progression or apoptosis. The present invention addresses such a need.